PROJECT SUMMARY/ABSTRACT: Electrical brain stimulation (EBS) is a FDA-approved neuromodulation therapy applied to several neurological disorders. However, the molecular basis of its efficacy remains unclear. Here we propose investigation of a glial mechanism for EBS mediated by astrocytes-derived extracellular vesicles (EVs). We recently discovered from both in vitro and in vivo experiments that electrical stimulation affects the release of EVs from astrocytes. In this proposal we will address two questions: 1) what is the molecular mechanism of electrical stimulation induced EVs release; 2) what is the biological function of the EVs released under electrical stimulations. Our exploratory research plan includes the following three steps: First - molecular characterization of astrocytic EVs (Aim1). In this aim we start from primary cultured astrocytes and systematically evaluate the effect of electrical stimulation parameters on EV cargos; Second - molecular mechanisms of how stimulation affects astrocytic EVs (Aim 2). In this aim we apply a high-resolution imaging technique to primary cultured astrocytes and focus on the trafficking of intracellular vesicles, including vesicle fusion to or budding off the plasma membrane. Third - functional characterizations of astrocytic EVs (Aim 3). In this aim, we will subject purified EVs collected in step 1 to both in vitro and in vivo functional testing. Focusing on neuronal activities as readout, we will first examine EV functions on primary cultured neurons; then we use in vivo animal models combined with 2-photon microscope technology to examine how EVs affect neuronal activities in both short- and long-term periods, and also how EVs affect animal behavior. In summary, our findings will help guide optimization of stimulation with next-generation EBS devices, with the ultimate goal of enhancing efficacy and treatments for patients with neurological disorders.